Compositions and methods for regulating receptor clustering

ABSTRACT

The invention relates to isolated complexes comprising one or more galectin associated with Mgat5 modified glycan or polylactosamine modified glycan, and isolated lectin-Mgt5 modified glycan lattice comprising an array of multivalent interactions among lectins, Mgat5 modified glycans, polylactosamine modified glycans, and/or glycoproteins. Methods for evaluating a test compound for its ability to regulate receptor clustering through glycans on cell surfaces; and methods for regulating receptor clustering on cell surfaces comprising altering glycans on the cell surface associated with receptor clustering are also discovered.

FIELD OF THE INVENTION

The invention relates to complexes, lattices, compositions and methodsfor regulating receptor clustering on cell surfaces.

BACKGROUND OF THE INVENTION

N- and O-linked glycans are found on both cell-surface and secretedproteins, many of which control proliferation and cell fate decisions inanimals. Tissue-specific expression of glycosyltransferases is asignificant factor controlling the glycan profiles observed indifferentiated cells (Paulson, Jc and Colley K J, J Biol Chem 1989,264:17615-17618). In addition, many glycosyltransferases compete foracceptor intermediates causing bifurcations of the pathways andadditional structural complexity (Schachter H, Biochem Cell Biol 1986,64:163-181).

Specific glycan structures regulate lymphocyte adhesion, re-circulationand maturation as demonstrated by the GDP-fucose deficiency in LADIIpatients (11), and immune defects associated with C2 GlcNAc-T(L) (12) orST3Gal-I (13) mutant mice. Depletion of theβ1,6N-acetylglucosaminyltransferase V (Mgat5) modified glycans byswainsonine, an inhibitor of α-mannosidase II, potentiatesantigen-dependent T cell proliferation, however, the molecular basis ofthis effect is unknown (14). Mgat5 catalyzes the addition of β1,6GlcNActo N-glycan intermediates found on newly synthesized glycoproteinstransiting the medial Golgi (15) (FIG. 1A). The glycans are elongated intrans-Golgi to produce tri (2,2,6) and tetra (2,4,2,6) antennaryN-glycans which are preferentially extended with N-acetyllactosamine(Galβ1GlcNAc) and polymeric forms of N-acetyllactosamine also known aspolylactosamine (6).

SUMMARY OF THE INVENTION

Applicants have demonstrated that differential receptor glycosylationeffects ligand-dependent clustering of receptors. Applicants illustratedthe effects of differential receptor glycosylation with T cell receptors(TCR). T cell activation requires clustering of a threshold number of Tcell receptors (TCR) at the site of antigen presentation, a number thatis reduced by CD28 co-receptor recruitment of signaling proteins to TCR(1-5). Applicants demonstrate that a deficiency inβ1,6N-acetylglucosaminyltransferase V (Mgat5), an enzyme in theN-glycosylation pathway, lowers T cell activation thresholds by directlyenhancing TCR clustering. Mgat5-deficient mice displayed kidneyautoimmune disease, enhanced delayed type hypersensitivity, andincreased susceptibility to experimental autoimmune encephalomyelitis.Thus, dysregulation of Mgat5 in humans may increase susceptibility toautoimmune diseases such as multiple sclerosis.

Recruitment of TCR to agonist-coated beads, TCR signaling, actinmicrofilament reorganization and agonist-induced proliferation wereenhanced in Mgat5^(−/−) T cells. Mgat5 initiates GlcNAc β1,6 branchingon N-glycans, thereby increasing N-acetyllactosamine (6), the lectin forgalectins (7, 8) proteins known to modulate T cell proliferation andapoptosis (9, 10). Indeed, galectin-3 was associated with the TCRcomplex at the cell surface, an interaction dependent on Mgat5.Pre-treatment of wild type T cells with lactose to compete for galectinbinding produced a phenocopy of Mgat5^(−/−) TCR clustering. These dataindicate that a galectin-glycoprotein lattice strengthened byMgat5-modified glycans restricts TCR recruitment to the site of antigenpresentation.

In accordance with an aspect of the invention an isolated complex isprovided comprising one or more lectin (e.g. a galectin) associated orinteracting with a Mgat5 modified glycan or polylactosamine modifiedglycan that is associated with receptor clustering. The invention alsoprovides a peptide derived from the binding domain of a lectin,preferably a galectin, that interacts with a Mgat5 modified glycan, or apolylactosamine modified glycan; and, an oligosaccharide derived from aMgat5 modified glycan or a polylactosamine modified glycan thatinteracts with one or more lectin (e.g. a galectin). The invention alsocontemplates antibodies specific for these complexes, peptides, andoligosaccharides.

The invention also contemplates an isolated lectin-Mgat5 modified glycanlattice comprising an array of multivalent interactions among lectins,Mgat5 modified glycans, polylactosamine modified glycans, and/orglycoproteins that are associated with receptor clustering. The Mgt5modified glycans and polylactosamine modified glycans are preferablypart of glycoproteins of receptors including but not limited to TCR,growth factor receptors, and cytokine receptors.

Still farther the invention provides a method for evaluating a testcompound for its ability to regulate receptor clustering through glycanson cell surfaces (e.g. through Mgat 5 modified glycans and/orpolylactosamine modified glycans) comprising assaying for alterations ofthe glycans in the presence of the test compound. Alterations of theglycans may increase or enhance, or inhibit or decrease receptorclustering thereby modifying signal transduction by the receptors.

In an aspect of the invention, a method is provided for evaluating atest compound for its ability to regulate receptor clustering through alectin-Mgat5 modified glycan lattice, in particular a galectin-Mgat5modified glycan lattice comprising determining the effect of the testcompound on the lattice or a component thereof. A test compound may be asubstance that interacts with a component of a lectin-Mgat5 modifiedglycan lattice. In particular, the substance may interact with a lectin(e.g. galectin), Mgat5 modified glycan, or polylactosamine modifiedglycan. The substance may be a molecule derived from a lectin (e.g.galectin), Mgat3 modified glycan, polylactosamine modified glycan, orlectin-Mgat5 modified glycan lattice; or, a substance which inhibits orenhances the interaction of a lectin (e.g. galectin) and a component ofa lectin −Mgat5 modified glycan lattice (e.g. the interaction of agalectin and Mgat5 modified glycan and/or polylactosamine modifiedglycan).

In an embodiment, the method comprises (a) mixing a galectin-Mgat5modified glycan lattice, or a galectin and one or more of a Mgat5modified glycan and a polylactosamine modified glycan, and a testcompound, under conditions which maintain the lattice or permit theformation of complexes between the galectin and one or more glycan; and(b) removing and/or detecting galectin-Mgat5 modified glycan lattice,complexes, galectin, Mgat5 modified glycan, or polylactosamine modifiedglycan.

The invention also encompasses the compounds identified using methods ofthe invention.

The invention also contemplates cell-based assays. In an aspect of theinvention, a method is provided comprising (a) providing cells withreceptors whereby clustering of the receptors is regulated through alectin-Mgat5 modified glycan lattice or a component thereof; (b) mixingthe cells, lectin, and a test compound under conditions which permit theformation of a lectin-Mgat5 modified glycan lattice, complexes between alectin and one or more glycan of the lattice, and/or receptorclustering; (c) detecting a lectin-Mgat5 modified glycan lattice,complexes, lectin, Mgat5 modified glycan, polylactosamine modifiedglycan, alterations to the lattice, complexes, lectin, Mgat5 modifiedglycan, or polylactosamine modified glycan, or receptor clustering; and(d) comparing to a control to determine if the test compound alters thelectin-Mgat5 modified glycan lattice or component thereof andpotentially regulates receptor clustering.

Differential glycosylation of receptors has been found to alter receptorclustering. Receptor clustering or oligomerization is a requisite eventfor signal transduction of receptors, including but not limited toreceptors that stimulate immune reactions, growth factor receptors, andcytokine receptors. Thus, differences in glycans (e.g. Mgat5 modifiedglycans or polylactosame modified glycans) on cell surfaces that areassociated with clustering of receptors including receptors thatstimulate immune reactions (e.g. T cell receptors, Ig receptors, B cellreceptors, NK receptors), the HER family of transmembrane receptortyrosine kinases [e.g. epidermal growth factor (EGF) receptor also knownas HER1 or Erb1, HER2 (neu, Erb2), HER3 (Erb3), and HER4 (Erb4)],cadherin receptors (e.g. E-cadherin and N-cadherin), interleukin (IL)receptors including IL-2 receptor, TNFγ receptor, and integrins, mayaffect clustering or oligomerization of these receptors.

The invention provides a method for regulating receptor clustering oncell surfaces comprising altering glycans on the cell surface associatedwith receptor clustering. In an aspect the invention provides a methodfor activating signal transduction in a cell with receptors that clusteror oligomerize to thereby initiate signal transduction comprisingaltering glycans associated with clustering or oligomerization of thereceptors.

Glycans can be altered by modulating one or more glycosyltransferaseenzyme involved in the synthesis of the glycans, in particular N-glycansand N-glycan intermediates. Altering glycans may involve increasing ordecreasing Mgat5 modified glycans or polylactosamine modified glycansassociated with clustering of the receptors. In a preferred embodiment,an enzyme involved in the synthesis of the glycans is modulated (e.g.Mgat5).

In accordance with the present invention, a method is provided forregulating receptor clustering on cell surfaces, in particularligand-dependent receptor clustering, more particularly T cell receptorclustering, comprising modulating Mgat5 activity, the amount of Mgat5modified glycans, polylactosamine modified glycans, or lectin-Mgat5modified glycan lattice, or the amount of binding or interaction of oneor more components of a lectin-Mgat5 modified glycan lattice, (e.g.Mgat5 modified glycans or polylactosamine modified glycans with lectinsthat bind to the glycans, for example, galectins).

In accordance with another aspect of the invention, a method is providedfor treating or preventing a condition associated with decreased orincreased receptor clustering or a receptor clustering defect in asubject comprising altering glycans associated with receptor clustering.Glycans can be altered by modulating a glycosyltransferase enzyme (e.g.Mgat5) involved in the synthesis of the glycans.

In accordance with a particular aspect of the invention, a method isprovided for treating or preventing a condition associated withdecreased or increased receptor clustering (more particularly T cellreceptor clustering), or a receptor clustering defect (more particularlya T cell receptor clustering defect), comprising modulating Mgat5activity, the amount of Mgat5 modified glycans, polylactosamine modifiedglycans, or lectin-Mgat5 modified glycan lattice, and/or the amount ofbinding or interaction of one or more components of a lectin-MgatVmodified glycan lattice (e.g. a galectin, a Mgat5 modified glycan,polylactosamine modified glycan, or glycoproteins).

The invention also contemplates compounds for regulating receptorclustering. The compounds may be capable of directly or indirectlymodifying glycans involved in receptor clustering. Such compounds maymodulate the activity of an enzyme involved in the synthesis of theglycans (e.g. a glycosyltransferase such as Mgat5), the amount of theglycans, (e.g. the amount of Mgat5 modified glycans or polylactosamineglycans), and/or the amount of binding of the glycans with a substancethat binds to the glycans thereby regulating receptor clustering (e.g.the binding or interaction of Mgat5 modified glycans and galectins). Theinvention also provides methods for assaying for such compounds.Compositions comprising such compounds are also within the scope of theinvention.

In accordance with an aspect of the invention there is provided a methodof, and products for, diagnosing and monitoring conditions characterizedby an abnormality in clustering of a receptor comprising assaying fordifferential glycosylation of the receptor. Differential glycosylationmay be assayed by determining the presence of Mgat5 modified glycans,polylactosamine modified glycans, lectin-Mgat5 modified glycan lattice,or an alteration or change in such glycans or lattice, compared to acontrol.

The invention relates to the control of glycan-lectin combinations (e.g.glacetin-polylactosamine modified glycan lattice) identified using theinvention.

These and other aspects, features, and advantages of the presentinvention should be apparent to those skilled in the art from thefollowing drawings and detailed description.

DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 Immune phenotype in Mgat5^(−/−) mice (A) Schematic of the GolgiN-glycan biosynthesis pathway shows Mgat5 (TV) in the production of atetra (2,4,2,6) antennary (numbers in brackets refer to the linkages ofthe antennae left to right). Abbreviations areoligosaccharyltransferase, OT; the α-glucosidases, GI, GII; theβ-N-acetylglycosaminyltransferases, TI, TII, TIV, TV T(i); theα1,2mannosidases, MI, α1,3/6mannosidases MII, MIII:β1,4-galactosyltransferases, Gal-T; α-sialyltransferases, ST; SW,position of swainsonine block. The boxed structureGalβ1,4GlcNAcβ1,6(Galβ1,4GlcNAcβ1,2)Manα binds L-PHA. The galectinbinding disaccharide N-acetyllactosamine (Galβ1,4GlcNAc) is present inall antennae, and units are marked with red brackets in polylactosamine.(B) Distribution of CD4+ and CD8+ cells in spleen and thymus by FACSanalysis using FITC- or phycoerythrin (PE)-conjugated antibodies(Pharmingen) reactive to CD3ε, CD4, and CD8. (C) TCR complex staining ofspleen cells by FITC-anti-CD3ε antibodies and FACS analysis. (D) Lightmicroscopy of kidney showing cresentic glomerulonephritis with a largecrescent (CR) of mononuclear cells and fibrin obliterating the Bowman'sspace (BS) in Mgat5^(−/−) mice. (E) DTH inflammatory response inMgat5^(−/−) (●) and Mgat5^(+/+) (□) mice exposed to oxazolone first ontheir back, then 4 days later on the right ear. The results are plottedas mean change ±S.E. in ear thickness relative to the vehicle-treatedleft ear for 7 Mgat5^(−/−) and 6 Mgat5^(+/+) control littermates. P<0.01with a student t test comparing the genotypes at 2-5 days.

FIG. 2 Mgat5^(−/−) T cells are hypersensitive to TCR agonists. (A)Spleen cells were cultured with anti TCRα/β antibodies for 48 h. Filledcircles, Mgat5^(−/−); open squares, Mgat5^(+/+). (B) Purified T cellsfrom spleen were stimulated for 48 h with anti-CD3ε antibody in theabsence (◯,□) or presence (●, ▪) of anti-CD28 antibody; Mgat5^(−/−)(circles) and Mgat5^(+/+) (squares) cells. (C) The Hill slope (n_(H)) ofthe sigmodal curves was calculated using Y=x^(nH)/(k^(nH)+x^(nH)). (D)Stimulation of splenic T cells with PMA plus ionomycin for 48 h. (E)Stimulation of splenic T cells from Mgat5^(−/−) (●) and Mgat5^(−/+) (□)mice with L-PHA and (F) Stimulation of splenic B cells with anti-IgMantibody for 48 h. The means±SD of triplicate determinations weregraphed.

FIG. 3 TCR clustering, actin reorganization and signaling in T cellsfrom Mgat5^(−/−) and Mgat5^(+/+) mice. (A) TCR and actin microfilamentdistribution in T cells stimulated by anti-CD3ε coated beads. (B) Mergedimages of Mgat5^(−/−) and Mgat5^(+/+) cells. Clustering was observed in5/6 and 0/6 randomly photographed cells, respectively. (C) TCRinternalization was monitored by FACS analysis using FITC-anti-TCR_(α/β)antibodies to measure cell surface TCR remaining at various times afterthe addition of anti-CD3ε antibody. Changes in mean fluorescenceintensity (MFI) with time are graphed. T cells from Mgat5^(−/−) (●,♦) orMgat5^(+/+) (□,*) mice were treated with anti-CD3ε antibody (●,□); orwith PMA (♦,* ). Similar results were obtained when the stimulation anddetection roles of anti-TCR_(α/β) and anti-CD3 were reversed. (D) Actinpolymer content in T cells from Mgat5^(−/−) (●), or Mgat5^(+/+) (□) miceat times after stimulation with anti-CD3ε antibody, measured by FACS.Western blot for phospho-Akt/PKB in T cell lysates following addition ofanti-CDε antibody is shown. The values below are fold increase in PKB-Pnormalized to PKB protein. (E) Ca²⁺ mobilization in purified T cellsfrom Mgat5^(−/−) (≡), and Mgat5^(+/+) (□) mice stimulated with anti-CD3εantibody. (F) Western blot with anti-phosphotyrosine antibody detectingphosphorylated proteins in T cells lysates after incubation withanti-CD3ε antibody coated beads for various times. A longer exposure wasused to reveal bands (arrowheads at left) migrating as p95 and p36 shownbelow. Arrows at the right indicate the positions of molecular massmarkers. (G) Immunoprecipitation of Zap70 and Western blotting forphosphotyrosine (pY) to detect Zap70 and CD3ζ (values below are CD3ζratio P23/P21).

FIG. 4 Lactose stimulates TCR aggregation and signaling in Mgat5^(+/+).Purified T cells pre-incubated for 20 min with buffer (A), 2 mM sucrose(B), or 2 mM lactose (C) then stimulated with anti-CD3ε antibody-coatedbeads for 10 min, and stained for TCR Enhanced TCR clustering wasobserved in 0/10, 1/10 and 9/10, respectively. (D) Mgat5^(+/+) T cellsincubated with increasing concentrations of disaccharide (1/3 serialdilution from 2.4 mM) and stimulated for 1 min with anti-CD3εantibody-coated beads were compared for phosphotyrosine. Arrows at theright indicate the positions of molecular mass markers. A longerexposure of the lower molecular weight portion of the blot is shown. (E)Galectin-3 detected by surface labeling with NHS-biotin on T cells.Below, association of galectin-3 with CD3ε and TCRα chain, and itsdisruption by Mgat5 deficiency and lactose is shown. (F) A modeldepicting restricted mobility of TCR by interaction with agalectin—glycoprotein network, which is stronger in Mgat5-expressingcells. (G) LacZ activity in untreated (white) and anti-CD3 and anti-CD28stimulated (grey) T cells from Mgat5^(−/−) mice. (H) L-PHA binding toMgat5^(+/+) T lymphocytes either untreated (white) or stimulated withanti-CD3 and anti-CD28 for 48 h (grey).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employedconventional biochemistry, enzymology, molecular biology, microbiology,and recombinant DNA techniques within the skill of the art. Suchtechniques are explained fully in the literature. See for example,Sambrook, Fritsch, & Maniatis, Molecular Cloning: A Laboratory Manual,Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984);Nucleic Acid Hybridization B. D. Hames & S. J. Higgins eds. (1985);Transcription and Translation B. D. Hames & S. J. Higgins eds (1984);Animal Cell Culture R. Freshney, ed. (1986); Immobilized Cells andenzymes IRL Press, (1986); and B. Perbal, A Practical Guide to MolecularCloning (1984).

Complexes, Peptides, and Oligosaccharides

In accordance with an aspect of the invention an isolated complex isprovided comprising one or more lectin associated or interacting with aMgat5 modified glycan, or polylactosamine modified glycan that isassociated with receptor clustering.

The term “isolated complex” refers to a complex substantially free ofcellular material or culture medium when produced in vitro, or chemicalreactants, or other chemicals when chemically synthesized.

“Lectin” refers to a molecule that interacts with, binds, or crosslinkscarbohydrates. Preferably a lectin employed in the present inventioninteracts with, binds, or crosslinks Mgat5 modified glycanspolylactosamine modified glycans, and/or glycoproteins. In anembodiment, the lectin is a galactose-binding protein, preferably agalectin.

“Galectin” refers to a member of the galectin family ofbeta-galactoside-binding proteins (see “Galectins: A Family of Animalbeta-Galactoside-Binding Lectins” (1994) by S. H. Barondes, V.Castronovo, D. N. W. Cooper, R. D. Cummings, K. Drickamer, et al., InCell 76, 597-598). Galectins includes lectins that bind beta-galactosidecarbohydrate moieties in a thiol-dependent manner. (Reviewed in Hadari,Y. R. et al. (1998) J. Biol. Chem. 270:3447-3453.) Galectins are widelyexpressed and developmentally regulated. Galectins contain acharacteristic carbohydrate recognition domain (CRD). The CRD is about140 amino acids and contains several stretches of about 1-10 amino acidsthat are highly conserved among all galectins. Examples of galectins aregalectin-1 through −10. In preferred embodiments of the invention, thegalectin is galectin-3. Galectin-3 has one CRD, a short N-terminaldomain and an intervening proline, glycine and tyrosine-rich domainwhich consists of repeats of 7-10 conserved amino acids. A “galectin”may be a monomer, dimer, or tetramer, preferably a dimer.

“Glycosyltransferase” refers to an enzyme involved in the synthesis ofglycans, preferably the synthesis of N-glycans or O-glycans, morepreferably N-glycans, most preferably tri (2,2,6) and tetra (2,4,2,6)antennary N-glycans, which are preferentially extended withN-acetyllactosamine and polylactosamine (i.e. polylactosamine modifiedglycan). Examples of such glycosyltransferase enzymes are Mgat5, core 2GlcNAc transferase, GlcNAcT(i), and β1,4 galactosyltransferase. The term“glycosyltransferase” includes a wild type enzyme, or part thereof, or amutant, variant or homolog of such an enzyme.

“Mgat5” refers to β1,6N-acetylglucosaminyltransferase V enzymes,preferably mammalian enzymes, that catalyze the addition ofN-acetylglucosamine in beta 1-6 linkage to the alpha-linked mannose ofbiantennary N-linked oligosaccharides. Examples of Mgat5 enzymes arefound on the ExPASy proteomics server as Enzyme: 2.4.1.155, and includehuman Mgat5 (Saito et al, 1994; gb:d17716, sw:q09328), and rat Mgat5(Shoreibah et al 1993, J. Biol. Chem. 268: 15381-15385; gb114284,sw:q08834). “Mgat5” includes the wild type enzyme, or part thereof, or amutant, variant or homolog of such an enzyme.

The term “wild type” refers to a polypeptide having a primary amino acidsequence which is identical with the native enzyme (for example, thehuman or mouse enzyme). The term “mutant” refers to a polypeptide havinga primary amino acid sequence which differs from the wild type sequenceby one or more amino acid additions, substitutions or deletions.Preferably, the mutant has at least 90% sequence identity with the wildtype sequence. Preferably, the mutant has 20 mutations or less over thewhole wild-type sequence. More preferably the mutant has 10 mutations orless, most preferably 5 mutations or less over the whole wild-typesequence.

The term “variant” refers to a naturally occurring polypeptide thatdiffers from a wild-type sequence. A variant may be found within thesame species (i.e. if there is more than one isoform of the enzyme) ormay be found within a different species. Preferably the variant has atleast 90% sequence identity with the wild type sequence. Preferably, thevariant has 20 mutations or less over the whole wild-type sequence. Morepreferably, the variant has 10 mutations or less, most preferably 5mutations or less over the whole wild-type sequence.

The term “part” indicates that the polypeptide comprises a fraction ofthe wild-type amino acid sequence. It may comprise one or more largecontiguous sections of sequence or a plurality of small sections. Thepolypeptide may also comprise other elements of sequence, for example,it may be a fusion protein with another protein (such as one which aidsisolation or crystallization of the polypeptide). Preferably thepolypeptide comprises at least 50%, more preferably at least 65%, mostpreferably at least 80% of the wild-type sequence.

The term “homolog” means a polypeptide having a degree of homology withthe wild-type amino acid sequence. The term “homology” refers to adegree of complementarity. There may be partial homology or completehomology. In an embodiment of the invention a glycostyltransferase, inparticular Mgat5, is substantially homologous to a wild type enzyme. Asequence that is “substantially homologous” refers to a partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid. Inhibition ofhybridization of a completely complementary sequence to the targetsequence may be examined using a hybridization assay (e.g. Southern ornorthern blot, solution hybridization, etc.) under conditions of reducedstringency. A sequence that is substantially homologous or ahybridization probe will compete for and inhibit the binding of acompletely homologous sequence to the target sequence under conditionsof reduced stringency. However, conditions of reduced stringency can besuch that non-specific binding is permitted, as reduced stringencyconditions require that the binding of two sequences to one another be aspecific (i.e., a selective) interaction. The absence of non-specificbinding may be tested using a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% homology oridentity). The substantially homologous sequence or probe will nothybridize to the second non-complementary target sequence in the absenceof non-specific binding.

A sequence of an enzyme contemplated by the invention may have at least60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity. The phrases“percent identity” or “% identity” refer to the percentage comparison oftwo or more amino acid or nucleic acid sequences. Percent identity canbe determined electronically using for example the MegAlign program(DNASTAR, Inc., Madison Wis.). The MegAlign program can createalignments between two or more sequences according to different methods,e.g., the Clustal method. (See, e.g., Higgins, D. G. and P. M. Sharp(1988) Gene 73:237-244.) Percent identity between nucleic acid sequencescan also be determined by other methods known in the art e.g., the JotunHein method. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)In addition, identity between sequences can be determined by othermethods known in the art, e.g., by varying hybridization conditions.

“Mgat5 modified glycan” refers to a GlcNAcβ1,6Manα1,6-branched N-glycanstructure. The glycans are produced by Mgat5 which catalyzes theaddition of P1,6GlcNAc to N-glycan intermediates found on newlysynthesized glycoproteins transiting the medial Golgi (15). The glycansare elongated in trans-Golgi to produce tri (2,2,6) and tetra (2,4,2,6)antennary N-glycans. A Mgat5 modified glycan may be substituted with forexample polylactosamine (i.e. it may be a polylactosamine modifiedglycan). A Mgat5 modified glycan may be part of or covalently linked toa cell surface glycoprotein, including a glycoprotein of the T cellreceptor complex.

“Polylactosamine modified glycan” refers to specific glycan structurescomprising N-acetyllactosamine (Galβ1,4GlcNAc) and polymeric forms ofN-acetyllactosamine, also known as poly N-acetyllactosamine orpolylactosamine (6). Preferably the polylactosamine modified glycan isan Mgat5 modified glycan substituted with poly N-acetyllactosamine. Apolylactosamine modified glycan may be part of or covalently linked to acell surface glycoprotein, including a glycoprotein of the T cellreceptor complex.

The invention also contemplates a lectin-Mgat5 modified glycan lattice,preferably a galectin-Mgat5 modified glycan lattice.

A “lattice” is an arrangement of multiple interacting molecules, inparticular, an arrangement or array of mulitvalent interactions amonglectins, glycans, and/or glycoproteins. A preferred lattice of theinvention is a lectin-Mgat5 modified glycan lattice.

A “lectin-Mgat5 modified glycan lattice” refers to a lattice formed fromthe multivalent interactions of lectins and Mgat5 modified glycans,polylactosamine modified glycans, and/or glycoproteins that areassociated with receptor clustering. When the lectin is a galectin thelattice is referred to as a “galectin-Mgat5 modified glycoproteinlattice”. The stoichiometry of components of a lattice preferablyprovides optimal occupation of the lectin-glycan binding sites to createstrong interactions among the components of the lattice resulting in animpediment to receptor clustering.

A lectin-Mgat5 modified glycan lattice, in particular a galectin-Mgat5modified glycan lattice may restrict clustering of receptors on cellsurfaces. By way of example, the galectin-Mgat modified glycan latticemay restrict TCR recruitment to the site of antigen presentation.

The invention also provides a peptide derived from the binding domain orbinding site of a lectin (e.g. a carbohydrate recognition domain of agalectin) that interacts with a glycan component of a lectin-Mgat5modified glycan lattice (e.g. a Mgat5 modified glycan, a polylactosaminemodified glycan, or glycoprotein); or, an oligosaccharide derived from aMgat5 modified glycan or polylactosamine modified glycan of alectin-Mgat5 modified glycan lattice that interacts with one or morelectin, in particular a galectin. The peptide may preferably be derivedfrom a carbohydrate recognition domain of a galectin.

The invention also relates to an oligosaccharide derived from a Mgat5modified glycan or polylactosamine modified glycan, preferably of a Tcell receptor, that interacts with one or more galectin.

By being “derived from” is meant any molecular entity which is identicalor substantially equivalent to the native binding site of a molecule ina complex, or lattice of the invention (e.g. a lectin in particular, agalectin, or a glycan, in particular a Mgat5 modified glycan orpolylactosamine modified glycan). A peptide or oligosaccharide derivedfrom a specific binding site may encompass the amino acid orcarbohydrate sequence of a naturally occurring binding site, any portionof that binding site, or other molecular entity that functions to bindto an associated or interacting molecule. A peptide or oligosaccharidederived from such a binding domain will interact directly or indirectlywith an associated or interacting molecule in such a way as to mimic thenative binding site. Such peptides and oligosaccharides may includecompetitive inhibitors, peptide mimetics, and the like.

The term “interact”, “interacting”, or “interaction” refers to a stableassociation between two molecules due to, for example, electrostatic,hydrophobic, ionic and/or hydrogen-bond interactions under physiologicalconditions.

“Peptide mimetics” are structures which serve as substitutes forpeptides in interactions between molecules (See Morgan et al (1989),Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimeticsinclude synthetic structures that may or may not contain amino acidsand/or peptide bonds but retain the structural and functional featuresof a peptide, or enhancer or inhibitor of the invention. Peptidemimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc.Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptidesof a designed length representing all possible sequences of amino acidscorresponding to a peptide, or enhancer or inhibitor of the invention.

The invention also contemplates an altered glycan of a cell surfaceglycoprotein associated with receptor clustering resulting from theinhibition of a glycosyltransferase involved in the synthesis of theglycan. In an embodiment the altered glycan is an altered Mgat5 modifiedglycan or an altered polylactosamine modified glycan. By way of example,an altered Mgat5 modified glycan has a deficiency of β1-6 branches, andan altered polylactosamine modified glycan has a deficiency ofN-acetyllactosamine or polylactosamine. An altered Mgat5 modified glycanor altered polylactosamine modified glycan cannot substantially interactor associate with a lectin, preferably a galectin.

Mgat5 modified glycans, polylactosoamine modified glycans and alteredglycans may be assayed using substances that bind to the glycans. Thesubstances that bind to the glycans may be antibodies or lectins. Forexample, leukoagglutinin (L-PHA) is a tetravalent plant lectin thatbinds specifically to Mgat5 modified glycans.

The invention contemplates antibodies specific for the complexes,lattice, peptides, oligosaccharides, and altered glycans of theinvention. Antibodies include intact monoclonal or polyclonalantibodies, and immunologically active fragments (e.g. a Fab, (Fab)₂fragment, or Fab expression library fragments and epitope-bindingfragments thereof), an antibody heavy chain, and antibody light chain, agenetically engineered single chain Fv molecule (Ladner et al, U.S. Pat.No. 4,946,778), humanized antibodies, or a chimeric antibody, forexample, an antibody which contains the binding specificity of a murineantibody, but in which the remaining portions are of human origin.Antibodies including monoclonal and polyclonal antibodies, fragments andchimeras, may be prepared using methods known to those skilled in theart.

Antibodies specific for a Mgat5 modified glycan, polylactosaminemodified glycan, complex, lattice, or an altered glycan may be producedin Mgat5^(−/−) mice using conventional methods.

Antibodies specific for the complexes, lattice, peptides,oligosaccharides, and altered glycans of the invention may be used todetect the complexes, lattice, etc. in tissues and to determine theirtissue distribution. In vitro and in suit detection methods using theantibodies of the invention may be used to assist in the prognosticand/or diagnostic evaluation of disorders mediated by or involvingreceptor clustering, more particularly T cell receptor mediateddisorders. Antibodies specific for the complexes, lattice, etc. of theinvention may also be used therapeutically to modulate receptorclustering, more particularly T cell receptor clustering (i.e. T cellactivation).

Evaluating Compounds that Regulate Receptor Clustering

The invention provides a method for evaluating a test compound for itsability to effect or regulate receptor clustering through glycans oncell surfaces (e.g. glycans of the receptor such as Mgat5 modifiedglycans or polylactosamine modified glycans). Changes to glycans on cellsurfaces may increase or decrease receptor clustering thereby modifyingsignal transduction by the receptors.

“Receptor clustering” or “clustering of receptors” refers to theassociation of one or more receptor molecules on the surface of a cellto thereby initiate signal transduction, endocytosis, and other eventsin the cell. Receptor clustering may be initiated or induced by theinteraction of ligands or anti-receptor antibodies with receptormolecules. Thus, in an aspect of the invention receptor clustering isligand-dependent. Examples of receptor molecules include but are notlimited to receptors that stimulate immune reactions (e.g. T cellreceptors, Ig receptors, B cell receptors, and NK receptors), members ofthe HER family of transmembrane receptor tyrosine kinases [e.g.epidermal growth factor (EGF) receptor also known as HER1 or Erb1, HER(neu, Erb2), HER3 (Erb3), and HER4 (Erb4)], cadherin receptors (e.g.E-cadherin and N-cadherin), interleukin (IL) receptors including IL-2receptor, TNFγ receptor, and integrins. In an embodiment, T cellreceptor clustering is down regulated by a lectin-Mgat5 modifed glycanlattice which slows the migration of T cell receptors into clusters atthe immune synapse. Dissociation of the lectin and glycan(s) of thelattice enhances T cell receptor clustering lowering the T cellactivation threshold.

An aspect of the invention provides a method for evaluating a testcompound for its ability to regulate receptor clustering through alectin-Mgat5 modified glycan lattice, in particular a galectin-Mgat5modified glycan lattice, or a component thereof.

Methods of the invention are designed to identify compounds orsubstances that affect receptor clustering particularly T cell receptorclustering (i.e. T cell activation). Novel substances are thereforecontemplated that bind to or interact with molecules in a complex orlattice, or bind to other molecules that interact with the molecules inthe complex or lattice, to compounds that interfere with, or enhance theinteraction of the molecules in a complex or lattice, or other compoundsthat interact with the molecules. Therefore, by way of example, acompound may be a substance that binds to a lectin (e.g. galectin), apolylactosamine modified glycan, or a Mgat5 modified glycan; a moleculederived from a lectin (e.g. galectin), Mgat5 modified glycan, orpolylactosamine modified glycan; or a substance which inhibits orenhances the interaction of a lectin (e.g. galectin) and a Mgat5modified glycan or a polylactosamine modified glycan.

A compound that enhances or inhibits the interaction of a lectin (e.g.galectin) and a Mgat5 modified glycan or polylactosamine modified glycanis intended to include a peptide or peptide fragment derived from thebinding site of a lectin (e.g. galectin), or oligosaccharide or fragmentthereof derived from the binding site of the Mgat5 modified glycan orpolylactosamine modified glycan. An enhancer or inhibitor will notinclude the full length sequence of the wild-type molecule. Peptidemimetics, oligosaccharide mimetics, and synthetic molecules withphysical structures designed to mimic structural features of particularpeptides or oligosaccharides, may serve as inhibitors or enhancers.Inhibitors or enhancers may affect receptor clustering, in particularT-cell receptor clustering. The enhancement or inhibition may be direct,or indirect, or b) a competitive or non-competitive mechanism.

The substances identified using the methods of the invention include butare not limited t peptides such as soluble peptides including Ig-tailedfusion peptides, members of random peptide libraries and combinatorialchemistry-derived molecular libraries made of D- and/or L-configurationamino acids, phosphopeptides (including members of random or partiallydegenerate, directed phosphopeptide libraries), oligosaccharides,antibodies [e.g. polyclonal, monoclonal, humanized, anti-idiotypic,chimeric, single chain antibodies, fragments, (e.g. Fab, F(ab)₂, and Fabexpression library fragments, and epitope-binding fragments thereof)],and small organic or inorganic molecules. The substance or compound maybe an endogenous physiological compound or it may be a natural orsynthetic compound.

The invention particularly contemplates a method for evaluating acompound for its ability to modulate the biological activity of acomplex or lattice of the invention, by assaying for an agonist orantagonist (i.e. enhancer or inhibitor) of the binding or interaction ofmolecules in the complex or lattice. The basic method for evaluating ifa compound is an agonist or antagonist of the binding of molecules in acomplex or lattice of the invention, is to prepare a reaction mixturecontaining the molecules and the substance under conditions which permitthe formation of complexes or a lattice, in the presence of a testcompound. The test compound may be initially added to the mixture, ormay be added subsequent to the addition of molecules. Control reactionmixtures without the test compound or with a placebo are also prepared.The formation of complexes or a lattice is detected and the formation ofcomplexes or a lattice in the control reaction but not in the reactionmixture indicates that the test compound interferes with the interactionof the molecules. The reactions may be carried out in the liquid phaseor the molecules, or test compound may be imnmobilized as describedherein.

It will be understood that the agonists and antagonists i.e. inhibitorsand enhancers that can be assayed using the methods of the invention mayact on one or more of the binding sites on the interacting molecules inthe complex or lattice including agonist binding sites, competitiveantagonist binding sites, non-competitive antagonist binding sites orallosteric sites.

The invention also makes it possible to screen for antagonists thatinhibit the effects of an agonist of the interaction of molecules in acomplex or lattice of the invention. Thus, the invention may be used toassay for a compound that competes for the same binding site of amolecule in a complex or lattice of the invention.

In an embodiment, the method comprises mixing a lectin-Mgat5 modifiedglycan lattice or a component thereof (e.g. lectin such as a galectin, aMgat5 modified glycan, polylactosamine modified glycan), and a testcompound under conditions which maintain the lattice or permit theformation of complexes between the lectin and one or more of the Mgat5modified glycan, and polylactosamine modified glycan, and removingand/or detecting lectin-Mgat5 modified glycan lattice, complexes,lectin, Mgat5 modified glycan, or polylactosamine modified glycan. Theinvention also encompasses the compounds identified using this method ofthe invention.

Substances which modulate the activity of a complex or lattice of theinvention can be identified based on their ability to bind to a moleculein a complex or lattice of the invention. Therefore, the invention alsoprovides methods for identifying novel substances which bind moleculesin a complex or lattice of the invention. Substances identified usingthe methods of the invention may be isolated, cloned and sequenced usingconventional techniques.

Novel substances which can bind with a molecule in a complex or latticeof the invention may be identified by reacting one of the molecules witha test substance which potentially binds to the molecule, underconditions which permit the formation of substance-molecule conjugatesand removing and/or detecting the conjugates. The conjugates can bedetected by assaying for substance-molecule conjugates, for freesubstance, or for non-complexed molecules. Conditions which permit theformation of substance-molecule conjugates may be selected having regardto factors such as the nature and amounts of the substance and themolecule.

The substance-molecule conjugate, free substance or non-complexedmolecules may be isolated by conventional isolation techniques, forexample, salting out, chromatography, electrophoresis, gel filtration,fractionation, absorption, polyacrylamide gel electrophoresis,agglutination, or combinations thereof. To facilitate the assay of thecomponents, antibody against the molecule or the substance, or labelledmolecule, or a labelled substance may be utilized. The antibodies,proteins, or substances may be labelled with a detectable substance asdescribed above.

A molecule, complex, or lattice of the invention, or a substance orcompound used in a method of the invention may be insolubilized. Forexample, a molecule, complex etc. may be bound to a suitable carriersuch as agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethylcellulose polystyrene, filter paper, ion-exchange resin, plastic film,plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acidcopolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon,silk, etc. The carrier may be in the shape of, for example, a tube, testplate, beads, disc, sphere etc. The insolubilized molecule, complex etc.may be prepared by reacting the material with a suitable insolublecarrier using known chemical or physical methods, for example, cyanogenbromide coupling.

Compounds that bind to molecules of a complex or lattice of theinvention or that interact with a molecule that binds to a molecule of acomplex or lattice of the invention may be assayed by identifyingprotein-protein or protein-carbohydrate interactions using conventionalmethods such as co-immunoprecipitation, crosslinking and co-purificationthrough gradients or chromatographic columns. Methods may also beemployed that result in the simultaneous identification of genes whichencode proteins interacting with a molecule. These methods includeprobing expression libraries with labeled molecules. Additionally, x-raycrystallographic studies may be used as a means of evaluatinginteractions with substances and molecules. For example, purifiedrecombinant molecules in a complex of the invention when crystallized ina suitable form are amenable to detection of intra-molecularinteractions by x-ray crystallography. Spectroscopy may also be used todetect interactions and in particular, Q-TOF instrumentation may beused. Two-hybrid systems may also be used to detect protein interactionsice vivo.

It will also be appreciated that the complexes or lattices of theinvention may be reconstituted in vitro and the effect of a testsubstance may be evaluated in the reconstituted system.

The invention also contemplates cell based assays. In an aspect of theinvention, a method is provided comprising (a) providing cells withreceptors whereby clustering of the receptors is regulated through alectin-Mgat5 modified glycan lattice or component thereof (e.g. lectin,Mgat5 modified glycans, and polylactosamine modified glycans); (b)mixing the cells, lectin, and a test compound, under conditions whichpermit the formation of a lectin-Mgat5 modified glycan lattice,complexes between a lectin and one or more glycan of the lattice, and/orreceptor clustering; (c) detecting a lectin-Mgat5 modified glycanlattice, complexes, lectin, Mgat5 modified glycan, polylactosaminemodified glycan, alterations to the lattice, complexes, lectin, Mgat5modified glycan, or polylactosamine modified glycan, or detectingreceptor clustering; and (d) comparing to a control to determine if thetest compound potentially regulates receptor clustering.

In another aspect of the invention, a method is provided comprising (a)providing cells with receptors whereby clustering of the receptors isregulated through a lectin-Mgat5 modified glycan lattice or componentthereof (e.g. lectin, Mgat5 modified glycans, and polylactosaminemodified glycans); (b) mixing the cells, lectin, a test compound, and aligand for the receptor that induces receptor clustering, underconditions which permit receptor clustering; (c) detecting receptorclustering; and (d) comparing to a control to determine if the testcompound potentially regulates receptor clustering. The lattice on thecell surface may regulate the threshold cooperativity and dynamic rangeof ligand dependent responses.

In an embodiment of the invention a method is provided which comprises;

-   -   (a) mixing cells with T cell receptors comprising Mgat5 modified        glycans or polylactosamine modified glycans, one or more        galectin, and a test compound under conditions suitable for        producing a galectin-Mgat5 modified glycan lattice;    -   (b) assaying for a galectin-Mgat5 modified glycan lattice; and    -   (c) comparing to a control in the absence of the test compound        to determine if the test compound has the potential to regulate        receptor clustering.

In an embodiment of the invention a method is provided which comprises;

-   -   (a) mixing cells with T cell receptors and Mgat5 modified        glycans and/or polylactosamine modified glycans on their        surface, one or more galectin, and a test compound under        conditions suitable for producing receptor clustering of the T        cell receptors;    -   (b) assaying for T cell receptor clustering or T cell signaling        or activation;    -   (c) comparing to a control to determine if the test compound has        the potential for regulating receptor clustering.

In a further embodiment of the invention, a method is providing forevaluating a test compound for its potential to regulate receptorclustering comprising:

-   -   (a) mixing cells with T cell receptors and Mgat5 modified        glycans or polylactosamine modified glycans on their surface,        one or more galectin, and a test compound under conditions        suitable for producing a galectin-Mgat5 modified glycan lattice;    -   (b) assaying for Mgat5 modified glycans, polylactosamine        modified glycans, galectin, or a galectin-Mgat5 modified glycan        lattice, or alterations to the glycans or lattice;    -   (c) comparing to a control where an alteration to a Mgat5        modified glycan, polylactosamine modified glycan, galectin, or a        galectin-Mgat5 modified glycan lattice, indicates that the test        compound has potential to regulate receptor clustering.

In a still further embodiment of the invention, a method is providingfor evaluating a test compound for its potential to regulate receptorclustering comprising:

-   -   (a) mixing Mgat5^(−/−) T cells and a test compound under        conditions suitable for clustering of T cell receptors on the T        cells;    -   (b) assaying for T cell receptor clustering or T cell signaling;        and    -   (c) comparing to a control to determine if the test compound has        potential to regulate receptor clustering.

In a still further embodiment of the invention, a method is providingfor evaluating a test compound for its potential to regulate receptorclustering comprising:

-   -   (a) mixing Mgat5^(−/−) T cells, one or more galectin, and a test        compound under conditions suitable for producing a        galectin-Mgat5 modified glycan lattice;    -   (b) assaying for galectin-Mgat5 modified glycan lattice or T        cell receptor clustering or T cell signaling; and    -   (c) comparing to a control to determine if the test compound has        potential to regulate receptor clustering.

The methods for evaluating a test compound for potential to regulate Tcell receptor clustering may include an antigen presenting cell, or abead coated with an antigen or anti-TCR antibody may be used to induceor initiate T cell receptor clustering.

Mgat5 modified glycans, polylactosoamine modified glycans, andlectin-Mgat5 modified glycan lattices may be assayed in the methods ofthe invention using substances that bind to Mgat5 modified glycans,polylactosoamine modified glycans, or the lattices. The substances thatbind to the glycans and lattices may be antibodies or lectins. Forexample, leukoagglutinin (L-PHA) may be used to assay for Mgat5 modifiedglycans.

T cell receptor clustering or T cell signaling or activation may beassayed using the methods illustrated herein and other standard methodsknown to a skilled artisan.

The invention contemplates methods for assaying for compounds andsubstances that regulate receptor clustering by modulating the activityof one or more enzyme involved in the biosynthesis of Mgat5 modifiedglycans or polylactosamine modified glycans, in particular aglycosyltransferase, more particularly Mgat5. Examples of methods forscreening for substances that modulate the activity of such enzymes areillustrated herein for Mgat5. The invention also contemplates methodsfor screening for compounds and substances that modulate the amount ofMgat5 modified glycans or polylactosamine modified glycans.

Therefore, the invention provides methods for screening for substanceshaving potential pharmaceutical utility in the treatment and diagnosisof conditions associated with increased or decreased receptorclustering, particularly T cell receptor clustering. In an embodiment ofthe invention a method of assaying for a therapeutic is providedcomprising assaying for a substance that inhibits or stimulates theactivity of Mgat5. Substances that inhibit or stimulate Mgat5 activitymay be identified by reacting Mgat5 with an acceptor substrate and asugar donor in the presence of a substance suspected of inhibitingMgat5, under conditions whereby the Mgat5 is capable of transferring thesugar donor to the acceptor substrate to produce a sugar donor-acceptorsubstrate complex, and determining the effect of the substance byassaying for sugar donor-acceptor substrate complexes, unreacted Mgat5,unreacted sugar nucleotide donor or unreacted acceptor substrate.

Suitable acceptor substrates include a saccharide, oligosaccharides,polysaccharides, glycopeptides, glycoproteins, or glycolipids which areeither synthetic with linkers at the reducing end or naturally occurringstructures, for example, asialo-agalacto-fetuin glycopeptide. The sugardonor may be a nucleotide sugar, dolichol-phosphate-sugar ordolichol-pyrophosphate-oligosaccharide, for example, uridinediphospho-N-acetylglucosamine (UDP-GlcNAc), or derivatives or analogsthereof.

The Mgat5 may be obtained from commercial sources; it may be purifiedfrom immortalized cell lines such as small cell lung cancer cells suchas QG (Gu, J. et al. J. Biochem. 113, 111-116, 1993); or prepared byexpression of the gene encoding Mgat5 in host cells.

The acceptor substrate or sugar donor may be labeled with a detectablesubstance, and the interaction of the enzyme with the acceptor and sugardonor will give rise to a detectable change. The detectable change maybe calorimetric, photometric, radiometric, potentiometric, etc. Theactivity of Mgat5 may also be determined using methods based on HPLC(Koenderman et al., FEBS Lett. 222:42, 1987) or methods employedsynthetic oligosaccharide acceptors attached to hydrophobic aglycones(Palcic et al Glycoconjugate 5:49, 1988; and Pierce et al, Biochem.Biophys. Res. Comm. 146: 679, 1987).

The Mgat5 is reacted with the acceptor substrate and sugar donor at a pHand temperature and in the presence of a metal cofactor, usually adivalent cation like manganese, effective for the enzyme to transfer thesugar donor to the acceptor substrate, and where one of the componentsis labeled, to produce a detectable change. It is preferred to use abuffer with the acceptor substrate and sugar donor to maintain the pHwithin the pH range effective for the proteins. The buffer, acceptorsubstrate, and sugar donor may be used as an assay composition. Othercompounds such as EDTA and detergents may be added to the assaycomposition.

Substances that inhibit or stimulate Mgat5 activity may also be assayedby treating immortalized cells that express Mgat5 with a substancesuspected of inhibiting or stimulating Mgat5, and comparing themorphology of the cells with the morphology of the cells in the absenceof the substance and/or with immortalized cells that do not expressMgat5.

Still further, a substance that inhibits or stimulates Mgat5 activitymay also be identified by treating a cell that expresses Mgat5 with asubstance that is suspected of affecting Mgat5 activity, and assayingfor Mgat5-modified glycans or polylactosamine modified glycans on thesurface of the cell. Mgat5-modified glycans and polylactosamine modifiedglycans can be measured using methods described herein and known in theart. For example, cells expressing Mgat5-modified glycans may be treatedwith a substance suspected of inhibiting or stimulating Mgat5-modifiedglycans. A lectin such as L-PHA is then added and the amount of bindingcan be compared to control cells which have not been treated with thesubstance and/or which do not express Mgat5-modified glycans.

In another method of the invention, immortalized cells expressingMgat5-modified glycans may be treated with a substance suspected ofinhibiting or stimulating Mgat5. The cells can be treated with a lectinsuch as L-PHA and the sensitivity to the lectin can be compared withcontrols cells which have not been treated with the substance and/orwhich do not express Mgat5. Examples of immortalized cells which can beused in the method are immortalized lung epithelial cell lines such asCHO cells, MvlLu cells, MDAY-D2 lymphoma, and lectin-resistant variantsof these cell lines, which are transfected with a Mgat5 vector and MDCKcells. In the absence of an inhibitor the cells should show signs ofmorphologic transformation. In particular, morphologic transformation isevidenced by (a) fibroblastic morphology, spindle shape and pile up; (b)the cells are less adhesive to substratum; (c) there is less cell-cellcontact in monolayer culture; (d) there is reduced growth-factorrequirements for survival and proliferation; (e) the cells grow insoft-agar or other semi-solid medium; (f) there is a lack of contactinhibition and increased apoptosis in low-serum high density cultures;(g) there is enhanced cell motility; and, (h) there is invasion intoextracellular matrix and secretion of proteases. Substances whichinterfere with one or more of these phenotypes may be considered toinhibit Mgat5.

Substances which inhibit or stimulate transcription or translation of agene encoding a glycosyltransferse in particular Mgat5 may be identifiedby transfecting a cell with an expression vector comprising arecombinant molecule containing a nucleic acid sequence encoding theglycosyltransferase (e.g. Mgat5), the necessary elements for thetranscription or translation of the nucleic acid sequence and a reportergene, in the presence of a substance suspected of inhibiting orstimulating transcription or translation of the gene encoding theglycosyltransferase (e.g. Mgat5), and comparing the level of expressionof the glycosyltransferase (e.g. Mgat5) or the expression of the proteinencoded by the reporter gene with a control cell transfected with thenucleic acid molecule in the absence of the substance. The method can beused to identify transcription and translation inhibitors or stimulatorsof the gene encoding the glycosyltransferase (e.g. Mgat5).

The nucleic acid molecule encoding the glycosyltransferase may beconstructed having regard to the sequence of the glycosyltransferasegene (e.g. see Saito et al., 1994 supra for Mgat5 gene sequence) usingchemical synthesis and enzymatic ligation reactions following proceduresknown in the art.

Suitable transcription and translation elements may be derived from avariety of sources, including bacterial, fungal, viral, mammalian, orinsect genes. Selection of appropriate transcription and translationelements is dependent on the host cell chosen, and may be readilyaccomplished by one of ordinary skill in the art. Examples of suchelements include: a transcriptional promoter and enhancer, an RNApolymerase binding sequence, a ribosomal binding sequence, including atranslation initiation signal. Additionally, depending on the host cellchosen and the vector employed, other genetic elements, such as anorigin of replication, additional DNA restriction sites, enhancers, andsequences conferring inducibility of transcription may be incorporatedinto the expression vector. It will also be appreciated that thenecessary transcription and translation elements may be Supplied by thenative gene and/or its flanking sequences.

Examples of reporter genes are genes encoding a protein such asβ-galactosidase (e.g. lac Z), chloramphenicol, acetyl-transferase,firefly luciferase, or an immunoglobulin or portion thereof such as theFc portion of an immunoglobulin preferably IgG. Transcription of thereporter gene is monitored by changes in the concentration of thereporter protein such as β-galactosidase, chloramphenicolacetyltransferase, or firefly luciferase. This makes it possible tovisualize and assay for expression of recombinant molecules to determinethe effect of a substance on expression of the glycosyltransferase (e.g.Mgat5) gene.

Mammalian cells suitable for carrying out the present invention includeany malignant cells, for example, COS (e.g., ATCC No. CRL 1650 or 1651),BHK (e.g., ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCCNo. CCL 2), and 293 (ATCC No. 1573). Suitable expression vectors fordirecting expression in mammalian cells generally include a promoter.Common promoters include SV40, MMTV, metallothionein-1, adenovirus Ela,CMV, immediate early, immunoglobulin heavy chain promoter and enhancer,and RSV-LTR.

Protocols for the transfection of mammalian cells are well known in theart and include calcium phosphate mediated electroporation, retroviral,and protoplast fusion-mediated transfection (see Sambrook et al.,Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring HarborLaboratory Press, 1989).

An agent that modulates Mgat5 activity, the amount of Mgat5 modifiedglycans, or the amount of binding of MgatV modified glycans and lectins(e.g. galectins) may comprise a complex of a lectin (e.g. galectin)associated with a Mgat5 modified glycan and/or a polylactosaminemodified glycan, or a lectin-Mgat5 modified glycan lattice; a peptidederived from the binding domain of a lectin (e.g. galectin) thatinteracts with Mgat5 modified glycan or polylactosamine modified glycan;and/or an oligosaccharide derived from the Mgat5 modified glycan thatinteracts with a lectin (e.g. galectin).

The reagents suitable for applying the methods of the invention toevaluate substances and compounds that modulate receptor clustering,more particularly T cell receptor clustering, may be packaged intoconvenient kits providing the necessary materials packaged into suitablecontainers. The kits may also include suitable supports useful inperforming the methods of the invention.

Regulating Receptor Clustering

The invention provides a method for regulating receptor clustering oncell surfaces comprising altering glycans on the cell surface associatedwith receptor clustering, in particular altering receptor glycosylation.Preferably the receptors are those that comprise Mgat5 modified glycansor polylactosamine modified glycans. Examples of receptors includereceptors that stimulate immune reactions (e.g. T cell receptors, Igreceptors, B cell receptors, NK receptors), the HER family oftransmembrane receptor tyrosine kinases [e.g. epidermal growth factor(EGF) receptor also known as HER1 or Erb1, HER2 (neu, Erb2), HER3(Erb3), and HER4 (Erb4)], cadherin receptors (e.g. E-cadherin andN-cadherin), interleukin (IL) receptors including IL-2 receptor, TNFγreceptor, and integrins.

Glycosylation may be altered by modulating one or moreglycosyltransferase enzyme involved in the synthesis of glycans involvedin receptor clustering, in particular N-glycans and N-glycanintermediates (e.g. Mgat5 modified glycans or polylactosamine modifiedglycans). Altering glycosylation may involve increasing or decreasingMgat5 modified glycans or polylactosamine modified glycans associatedwith receptor clustering. In a preferred embodiment, an enzyme involvedin the synthesis of the glycans is modulated (e.g. Mgat5).

In accordance with a particular aspect, the present invention relates toa method for regulating receptor clustering on cell surfaces, inparticular ligand-dependent receptor clustering, more particularly Tcell receptor clustering comprising modulating Mgat5 activity, theamount of Mgat5 modified glycans, polylactosamine modified glycans, orlectin-Mgat5 modified glycan lattice, or the amount of binding orinteraction of one or more of MgatV modified glycans or polylactosaminemodified glycans and lectins that interact with the glycans (e.g.galectins).

Receptor clustering or oligomerization, in particular ligand-dependentreceptor clustering, may be reduced or inhibited by increasing theamount or levels of Mgat5 modified glycans, polylactosamine modifiedglycans, and/or lectin-Mgat5 modified glycan lattice, increasing theactivity or amount of one or more glycosyltransferase enzyme, orenhancing the interaction between glycans involved in receptorclustering and substances that bind to the glycans that regulatereceptor clustering (e.g. lectins).

Receptor clustering, in particular ligand-dependent receptor clustering,may be enhanced or increased and glycosylation of the receptor may bealtered by decreasing the amount or levels of Mgat5 modified glycans,polylactosamine modified glycans, and/or lectin-Mgat5 modified glycanlattice, decreasing the activity or amount of one or moreglycosyltransferase enzyme, or inhibiting the interaction betweenglycans involved in receptor clustering and substances that bind to theglycans that regulate receptor clustering.

In an aspect of the invention a method is provided for lowering T cellactivation threshold to agonists comprising decreasing Mgat5 modifiedglycans, polylactosamine modified glycans, or galectin-Mgat5 modifiedglycan lattice on the surface of the cells, or dissociating galectinfrom such glycans or lattice thereby lowering the T cell activationthreshold. Mgat5 modified glycans, polylactosamine modified glycans, orgalectin-Mgat5 modified glycan lattice on the surface of the cells maybe decreased by inhibiting a glycosyltransferase such as Mgat5.

In another aspect, a method is provided for restricting T cell receptorrecruitment in response to an agonist or increasing T cell activationthreshold comprising increasing Mgat5 modified glycans, polylactosaminemodified glycans, or galectin-Mgat5 modified glycan lattice on thesurface of the T cells, or enhancing the interaction between one or morecomponents of a galectin-Mgat5 modified glycan lattice (e.g. a galectinand glycans of the lattice) thereby increasing the T cell activationthreshold. The amount of glycans or lattice on the surface of the cellsmay be increased by increasing the levels or activity of one or moreglycosyltransferase enzyme (e.g. Mgat5).

In accordance with another aspect of the invention, a method is providedfor treating or preventing a condition associated with decreased orincreased receptor clustering or a receptor clustering defect in asubject comprising altering glycans associated with or involved inreceptor clustering. Glycans can be altered or modified by modulating aglycosyltransferase enzyme involved in the synthesis of the glycans.

In accordance with a particular aspect of the invention, a method isprovided for treating or preventing a condition associated withdecreased or increased receptor clustering, more particularly T cellreceptor clustering, comprising modulating Mgat5 activity, the amount ofMgat5 modified glycans, polylactosamine modified glycans, and/orlectin-Mgat5 modified glycan lattice, and/or the amount of binding orinteraction of one or more MgatV modified glycans, polylactosaminemodified glycans and lectins e.g. galectins.

A receptor clustering defect may be involved in conditions such asautoimmune diseases or proliferative disorders such as cancer.

A condition associated with increased T cell receptor clustering mayinclude a T cell mediated autoimmune disease such as insulin-dependentdiabetes mellitus, multiple sclerosis, rheumatoid arthritis, myastheniagravis, systemic lupus erythematosus, autoimmune hemolytic anemia,glomerulonephritis, enhanced delayed type hypersensitivity, allergicconditions, hypersensitivity, and autoimmune encephalomyelitis.Conversely, T cell recognition of cancers and immune therapy of canceris limited by weak stimulation of T cells by tumor cells. The presentinvention may also be used to treat cancers susceptible to immunemodulation.

In an aspect the invention contemplates a method for treating orpreventing an autoimmune disease in a subject comprising reducing T cellreceptor clustering in the subject T cell receptor clustering is reducedby increasing the amount of Mgat5 modified glycans, polylactosaminemodified glycans, and/or lectin-Mgat5 modified glycan lattice on thesurface of T cells of the subject In an embodiment, the method comprisesup regulating or increasing the amount of Mgat5.

In an aspect the invention contemplates a method for treating orpreventing cancer in a subject comprising increasing T cell receptorclustering in the subject T cell receptor clustering is increased bydecreasing the amount of Mgat5 modified glycans, polylactosaminemodified glycans, and/or lectin-Mgat5 modified glycan lattice on thesurface of T cells of the subject. In an embodiment, the methodcomprises down regulating or decreasing the amount of Mgat5.

In an embodiment of the invention, a method is provided for treating orpreventing a condition associated with a growth factor receptor, inparticular epidermal growth factor receptor, comprising regulatingclustering or oligomerization (e.g. dimerization) of the growth factorreceptor by altering glycosylation of the receptor, modulating Mgat5activity, the amount of MgatV modified glycans, polylactosamine modifiedglycans, and/or the binding of MgatV modified glycans or polylactosaminemodified glycans and lectins for the glycans. Inhibition of growthfactor receptor clustering may be useful in treating conditionsinvolving aberrant grouch factors including but not limited to cancerssuch as solid human cancers, NSCL, breast cancer, head and neck cancer,gastric cancer, prostate cancer, bladder cancer, ovarian cancer,colorectal cancer, gliobastomas, and renal cell carcinoma One or moreagents may be used to regulate receptor clustering. In particular, oneor more agents may be used to modulate glycosyltransferase activity,more particularly Mgat5 activity, the amount of Mgat5 modified glycansor polylactosamine glycans, the amount of binding of MgatV modifiedglycans or polylactosamine modified glycans and lectins that interactwith the glycans (e.g. galectins), or the amount of lectin-Mgat5modified glycan lattice.

Agents that modulate glycosyltransferase activity, more particularlyMgat5 activity, include known inhibitors or enhancers ofglycosyltransferases, compounds or substances identified using themethods described herein, nucleic acids encoding theglycosyltransferases, and antisense sequences of the nucleic acidsequence encoding the glycosyltransferases. Examples ofglycosyltransferase inhibitors and enhancers are illustrated herein forMgat5.

By way of example, known inhibitors of Mgat5 include an analog of theacceptor substrate for Mgat5, βGlcNAc (1,2)αMan(1,6)βManOR, where thereactive 6′OH group has been removed (Palcic, M. M. et al., J. Biol.Chem. 265 (12) 6759-6769). Inhibitors of enzymes earlier on in the Golgioligosaccharide processing pathway may also be used to inhibit Mgat5activity. Examples of inhibitors of other enzymes in the Golgioligosaccharide processing pathway include mannosidase inhibitors suchas swainsonine, 1,5-dideoxy-1,5-imino-5-mannitol and1,4-dideoxy-1,4imino-D-mannitol.

Recombinant molecules containing the nucleic acid sequence Mgat5 in anantisense orientation may be used to inhibit Mgat5 activity. The nucleicacid sequence shown in Saito, H. et al. Biochem. Biophys. Res. Comm.198;318-327, 1994, or parts thereof, may be inverted relative to theirnormal presentation for transcription to produce antisense nucleic acidmolecules. The antisense nucleic acid molecules may be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. The antisense nucleic acid molecules or parts thereof,may be chemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed with mRNA or the native gene e.g. phosphorothioatederivatives and acridine substituted nucleotides. The antisensesequences may be produced biologically using an expression vectorintroduced into cells in the form of a recombinant plasmid, phagemid orattenuated virus in which antisense sequences are produced under thecontrol of a high efficiency regulatory region, the activity of whichmay be determined by the cell type into which the vector is introduced.

The amount of Mgat5 modified glycans may be increased in cells byadministering Mgat5, a nucleic acid molecule encoding Mgat5, an agentthat stimulates Mgat5, or a complex of the invention. Increased cellsurface Mgat5 modified glycans may enhance the lectin-Mgat5 modifiedglycan lattice (e.g. galectin-Mgat5 modified glycan lattice) at the cellsurface so as to restrict receptor clustering (e.g. T cell receptorclustering). The amount of polylactosoamine modified glycans may beincreased in cells by administering one or more enzyme necessary for theproduction of the glycans, a nucleic acid molecule encoding the enzyme,an agent that stimulates the enzyme, or a complex of the invention.Increased cell surface polylactosamine modified glycans may enhance thelectin-Mgat5 modified glycan lattice (e.g. galectin-Mgat5 modifiedglycan lattice) at the cell surface so as to restrict receptorclustering (e.g. T cell receptor clustering). These approaches may beuseful in the prevention and treatment of T cell mediated autoimmunediseases.

Agents, compounds, and substances described herein or identified using amethod of the invention may be formulated into pharmaceuticalcompositions for administration to subjects in a biologically compatibleform suitable for administration in vivo. By “biologically compatibleform suitable for administration in vivo” is meant a form of thesubstance to be administered in which any toxic effects are outweighedby the therapeutic effects. The substances may be administered to livingorganisms including humans, and animals. Administration of atherapeutically active amount of the pharmaceutical compositions of thepresent invention is defined as an amount effective, at dosages and forperiods of time necessary to achieve the desired result For example, atherapeutically active amount of a substance may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of antibody to elicit a desired response inthe individual. Dosage regima may be adjusted to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The active substance may be administered in a convenient manner such asby injection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active substance may be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions that may inactivate the compound.

The compositions described herein can be prepared by per se knownmethods for the preparation of pharmaceutically acceptable compositionswhich can be administered to subjects, such that an effective quantityof the active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa, USA 1985). On this basis,the compositions include, albeit not exclusively, solutions of thesubstances or compounds in association with one or more pharmaceuticallyacceptable vehicles or diluents, and contained in buffered solutionswith a suitable pH and iso-osmotic with the physiological fluids.

The activity of a pharmaceutical composition, an agent, compound, orsubstance described herein or identified using a method described hereinmay be confirmed in animal experimental model systems.

In accordance with an aspect of the invention there is provided a methodof, and products for, diagnosing and monitoring conditions characterizedby an abnormality in receptor clustering comprising assaying fordifferential glycosylation of the receptor. Differential glycosylationmay be assayed by determining the presence of Mgat5 modified glycans,polylactosamine modified glycans, lectin-Mgat5 modified glycan lattice,or an alteration or change in such glycans or lattice, compared to acontrol.

In an embodiment, a method of, and products for, diagnosing andmonitoring conditions characterized by an abnormality or defect ofreceptor clustering involving the interaction of a galectin and Mgat5modified glycan or polylactosamine modified glycan is providedcomprising determining the presence of one or more of a complex of theinvention, a Mgat5 modified glycan, a polylactosamine modified glycan, agalectin-Mgat5 modified glycan lattice, or one or more of an alteredMgat5 modified glycan, polylactosamine modified glycan, or agalectin-Mgat5 modified glycan lattice.

Nucleic acid molecules encoding MgatV, an Mgat5 polypeptide, andantibodies specific for Mgat5, complexes, lattice, oligosaccharides, orpeptides of the invention may be used in the prognostic and diagnosticevaluation of conditions associated with increased or decreased receptorclustering more particularly T cell receptor clustering, and theidentification of subjects with a predisposition to such conditions. Inan embodiment, the nucleic acid molecules, Mgat5, and antibodies may beused in the diagnosis and staging of T cell mediated autoimmunediseases.

The following non-limiting example is illustrative of the presentinvention.

EXAMPLE

Methods

Delayed-type hypersensitivity (DTH) skin reaction: To inducedelayed-type hypersensitivity, 100 μl of 5% (w/v)4-ethoxymethilene-2-phenyl-2-oxazolin-5-one (oxazolone) (Sigma) inethanol/acetone (3:1, v/v) was injected epicutaneously to the shavedbacks of the 129/sv mice. Four days after sensitization, 25 μl of 1%,(w/v) oxazolone was applied on each side of the right ear, and the leftear received 25 μl of olive oil/acetone on each side. Ear swelling wasmeasured with a micrometer at 24 h intervals for the next 5 days, andswelling was reported as the difference between the ear thickness of theright minus the left ears.

EAE model: Mice (129/sv) 8-12 weeks of age were injected subcutaneouslywith 100 Ill of rabbit MBP (Sigma) emulsified 1:1 with complete Freundsadjuvant at three different total doses (25, 100 and 500 μg/mouse). Micewere observed from day 5 to day 50, and observations were done blindedwith respect to the genotype until day 36. For lower doses of 25 and 50μg/mouse, half the total was injected on day 0 in the right flank andthe other half on day 7 in the left flank. The high dose of 500 μg/mousewas injected all on day 0 at the base of the tail, and 500 μg ofpertussis toxin was injected via the tail vein on day 0 and day 2.

T cell proliferation: Naive T-cells were purified from spleens of 8-12week old mice by negative selection using CD3+T cell purificationcolumns (R&D laboratories) or by panning on plates pre-coated withanti-CD19 antibody (Pharmingen). T cell proliferation was measured byculturing cells for 48 h in RPMI, 10% FCS, 10⁻⁵ M 2-mercaptoethanol inthe presence of one or more of the following soluble antibodies: hamsteranti-mouse CD3ε (clone 2C11; Cedarlane), hamster anti-mouse TCRα/β(clone H59.72; Pharmingen) or 0.5 μg/ml anti-mouse CD-28 (Pharmingen).PMA at 10 ng/ml and ionomycin at 0.5 μg/ml were also used to stimulatecells. Two μCi of ³H-thymidine were added for the last 20 h ofincubation, and cells were harvested on fiberglass filters andradioactivity was measured in a β-counter.

TCR clustering: Six-micron polystyrene beads (Polysciences) in PBS werecoated with hamster anti-mouse CD3ε antibody (Clone 2C11; Cederlane) at2 μg/ml antibody followed by coating with 200 μg/ml bovine serum albumin(BSA). To measure TCR clustering, 5×10⁴ T cells were incubated with2.5×10⁵ anti-CD3ε antibody-coated beads in 100 μl RPMI 1640+10% FCS at37° C. for 10 minutes placed on poly-L-lysine coated cover slips. Thecells were fixed with 10% formalin, stained with 2 μg/ml fluoresceinisothiocyanate (FITC) labeled anti-TCRα/β antibody (Pharmigen),solubilized with 0.2% Triton X-100, labelled with rhodamine-phalloidinand Hoechst, and then visualized by deconvolution microscopy. Fordisaccharide competition, wild type T cells were incubated for 20 minwith 0, 0.01, 0.03, 0.09, 0.27, 0.8, and 2.4 mM of disaccharide prior toexposure to anti-CD3ε antibody beads. To measure TCR internalization,purified splenic T cells stimulated with either 0.1 μg/ml⁻¹ anti-CD3antibody or with 10 ng/ml PMA for varying lengths of time were harvestedand stained with FITC-anti-TCRα/β. PMA concentrations were not limitingas 10, 50 and 100 ng/ml produced similar internalization and cellactivation results. To measure actin reorganization, purified splenic Tcells, stimulated with 0.1 μg/ml⁻¹ anti-CD3 for varying lengths of time,were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS andstained with rhodamine-phalloidin and mean fluorescence intensity (MFI)was determined by FACS.

TCR signaling: T cells (1×10⁶) and anti-CD3ε antibody coated beads(5×10⁶ at 0.4 μg/ml antibody) in 100 μl RPMI 1640 were pelleted,incubated at 37° C. for various times, then solublized with ice cold 50mM Tris pH 7.2, 300 mM NaCl, 0.5% Triton X-100, protease inhibitorcocktail (Boeringer Mannheim) and 2 mM orthovanadate. Zap-70 wasimmunoprecipitated by incubating whole cell lysates with rabbitpolyclonal anti-Zap-70 agarose conjugate (Santa Cruz) overnight at 4°C., followed by one wash with lysis buffer and 3 washes with PBS.Western blotting was done with whole cell lysates or immunoprecipitatesseparated on SDS-PAGE gels under reducing conditions, transferredelectrophoretically onto PVDF membranes and immunoblotted withantibodies to Akt/PKB (NEB), phospho-Akt/PKB (NEB), phosphotyrosine(clone 4G10, Upstate Biotechnology), Zap 70 (clone Zap70-6F7, Zymed),TCRα (polyclonal, Santa cruz) and rabbit anti-galectin-3 (Dr. A Raz,University of Michigan). Cell surface proteins were biotinylated usingsulfosuccininmidobiotin (NHS-biotin) for 30 min, PBS pH 8.0. Cells werelysed and labeled protein was captured on streptoavidin-agarose beads.To cross-link surface proteins on purified naive T cells, thehomobifunctional cross-linker dithiobis(sulfosuccinimydylpropionate(DTSSP) was used at 0.1 mg/ml with 10⁶ cell/ml in PBS pH 8.0 for 10 minat 20° C. T cells were preincubated for 20 min with or without 2 mMlactose, and reacted with DTSSP in the presence of the same. Aliquots ofcell lysate were immunoprecipitated with rabbit anti-galectin-3 antibodyor non-immune rabbit serum (NS), separated on reducing— SDS-PAGE andWestern blotted for CD3ε and TCRα chain. The band above CD3ε iscross-reactivity of secondary antibody with light-chain.

To measure Ca⁺⁺ mobilization, purified T cells were loaded with 10 μM AMester of Fluo-3 (Molecular Probes) washed and stimulated with 10 μg/mlof anti-CD3ε antibody at 37° C. Emission at 525 nm was taken using aspectrofluorimeter with excitation at 488 nm. Data is plotted as afraction of the Ca⁺⁺ mobilized by addition of 2 μg/ml⁻¹ of ionomycin.LacZ activity in Mgat5^(−/−) T cells was detected by loading cells withflourescin-di-β-D-galactopyranoside (FDG) (Molecular Probes) at 10° C.,and allowing the reaction to proceed for 30 min. The reaction wasstopped by the addition of 1 mM phenyl-β-thiogalactoside.

Results and Discussion

To explore the role of Mgat5 in T cell immunity, Mgat5-deficient micewere examined for evidence of immune dysfunction. Mgat5^(−/−) mice areborn healthy, and lack Mgat5 N-glycan products in all tissues examined(16). At 3 months of age, peripheral white blood cells, erythrocyte andserum levels of IgM and IgG were comparable in Mgat5^(−/−), Mgat5^(+/−)and Mgat5^(+/+) mice (data not shown). The CD4 and CD8 reactive T cellpopulations in the spleen and thymus were also in the normal range(FIGS. 1B,C). At 12-20 months of age, an increased incidence ofleukocyte colonies in kidney and enlarged spleens were observed inMgat5^(−/−) mice. Furthermore, 32% of the Mgat5^(−/−) (6/19 mice) hadmacroscopic hematuria, mononuclear infiltrates and extensiveaccumulation of fibrin within Bowman's space (crescents), characteristicof proliferative glomerulonephritis (FIG. 1D). This form of renal injuryis often observed in autoimmune mediated glomerulonephritis. Milderrenal defects were observed in 68% of the Mgat5^(−/−) mice but not inthe Mgat5^(+/−) or Mgat5^(+/+) mice (0/19).

To examine T cell responses in the mice, a type IV delayed-typehypersensitivity (DTH) reaction was induced and tissue swelling wasmeasured. The protein-reactive hapten oxazolone was applied topically tothe backs of the mice, then again 4 days later to the right ear. Earswelling in Mgat5^(+/+) mice peaked 24 hours post application, andswelling was completely gone by day 5. Ear swelling in Mgat5^(−/−) miceattained a higher maximum between 48 and 72 h, and persisted for alonger time (FIG. 1E). To study T cell dependent autoimmunity in vivo(17), experimental autoimmune encephalomyelitis (EAE) was induced byimmunizing mice with myelin basic protein (MBP) at 3 doses: 25, 100 and500 μg/mouse. At the lowest dose of MBP, 25 μg/mouse, the incidence ofEAE was significantly greater in Mgat5 deficient mice. Furthermore, 25and 100 μg/mouse doses of MBP produced more severe EAE in Mgat5^(−/−)mice compared to wild type littermates, characterized by an earlieronset, greater motor weakness and more days with disease (Table 1).Myelin injections of 500 μg/mouse induced disease in all mice withgreater peak scores and no significant differences in disease incidenceor severity between genotypes. These results indicate that mice lackingMgat5-modified glycans are more susceptible to DTH and EAE autoimmunedisease.

In vitro, splenic T cells from Magt5−/− mice hyperproliferated inresponse to anti-TCRα/β antibody (FIG. 2A). To examine thishypersensitivity in more detail, purified ex vivo T cells were culturedat low density and stimulated with increasing concentrations of solubleanti-CD3ε antibody in the presence or absence of anti-CD28 antibody(FIG. 2B). Both the Mgat5 deficiency and CD28 engagement reduced therequirements for TCR agonist as indicated by D₅₀ values and wereadditive when combined (FIG. 2C). Furthermore, the apparent Hillcoefficient (n_(H)), a measure of synchrony in the responding cellpopulation, was increased by both the Mgat5 deficiency and by CD28engagement Therefore, the stimulatory effects of the Mgat5 mutation andCD28 co-receptor engagement were additive and similar in potency.

Alterations in cell surface TCR complex levels and intracellularsignaling potential of T cells were examined and ruled-out as possiblecauses of the Mgat5^(−/−) hypersensitivity. The Mgat5 deficiency did notsignificantly alter cell surface expression of CD3, CD4, CD8, TCRα/β,CD28 or CTLA-4 glycoproteins in resting T cells (FIGS. 1B, C and datanot shown). Intracellular signaling potential in Mgat5^(−/−) T cells isnormal, as treatment with the phorbol ester PMA and the Ca⁺⁺ ionophoreionomycin stimulated T cells equally well from mice of both genotypes(FIG. 2D).

The relationship between cell surface Mgat5-modified glycans and T cellactivation was examined. Leukoagglutinin (L-PHA) is a tetravalent plantlectin and commonly used T cell mitogen that binds specifically toMgat5-modified glycans. Mgat5^(−/−) T cells were completely unresponsiveto L-PHA, confirming that Mgat5-modified glycans are required forstimulation by this lectin (FIG. 2E). L-PHA reactive N-glycans are alsopresent on B cells, but L-PHA is not a B cell mitogen. Furthermore, Bcell responses to anti-IgM antibody, LPS and IL-4 plus anti-CD40antibody were similar for cells from Mgat5^(−/−) and Mgat5^(+/+) mice(FIG. 2F and data not shown). In T cells, L-PHA induces signaling commonto TCR engagement, including phosphorylation of CD3ζ, Ca⁺⁺ mobilization,PKC-γ and Ras/mitogen-activated protein kinase (Mapk) activation (18;19). The TCRα/β chains have 7 N-glycans in total, and some are branchedcomplex-type structures with L-PHA reactivity (20; 21). These dataindicate that Mgat5-modified glycans are present on glycoproteins of theTCR complex and required for L-PHA mitogenesis.

When bound to major histocompatibility complex (MHC)/peptide, TCRscluster with an inherent affinity greater than unligated TCR and thestability of these clusters is critical for intracellular signaling(22). However, the density of TCRs measured at the site of T cell-APC(antigen-presenting cell) contact is only marginally increased relativeto the remaining cell surface, leaving the majority of the TCRsunengaged by MHC/peptide (4). It is possible that ligand induced TCRclustering in the plane of the membrane may be increased in the absenceof Mgat5-modified glycans, thus lowering Mgat5^(−/−) T-cell activationthresholds. To visualize TCR reorganization in response to anantigen-presenting surface, polystyrene beads were coated with anti-CD3εantibody and incubated with purified ex vivo T cells. After 10 minutesof contact, TCRs in Mgat5^(−/−) cells was markedly more concentrated atthe bead surface compared to Mgat5^(+/+) cells (FIGS. 3A, B). TCRs onwild type cells could not be induced to cluster to the same extent asMgat5^(−/−) cells even with longer incubations (20 min) or usinganti-CD3ε plus anti-CD28 coated beads (data not shown). Actinmicrofilaments were more concentrated at the bead contact site inMgat5^(−/−) cells, and overlapped more extensively with TCR in themerged images compared to Mgat5^(+/+) T cells (FIGS. 3A, B). TCRs areinternalized following productive TCR clustering (1), and this wassignificantly greater in Mgat5 ^(−/−) compared to Mgat5^(+/+) cells(FIG. 3C, solid lines). Intracellular signaling mediated by PMAtreatment induces TCR internalization but at similar rates inMgat5^(−/−) and Mgat5^(+/+) cells (FIG. 3C, dotted lines). Microfilamentre-organization was more rapid in Mgat5 deficient T cells followingsoluble anti-CD3ε antibody stimulation (FIG. 3D). Akt/protein kinase B(PKB) phosphorylation is dependent upon phosphoinositide 3-OH kinaseactivity, which stimulates Rac/CDC42 GTPases and actin filamentre-organization (23). Phosphorylated Akt/PKB displayed a greater foldincrease in Mgat5^(−/−) compared to Mgat5^(+/+) T cells (FIG. 3D).Mobilization of intracellular Ca²⁺ following stimulation with solubleanti-CD3ε antibody was enhanced in the absence of Mgat5-modified glycans(FIG. 3E). Tyrosine phosphorylation of multiple proteins was increasedand persisted longer in Mgat5^(−/−) T cells exposed to anti-CD3εantibody coated beads. (FIG. 3F). Immunoprecipitation of Zap70 revealedincreased phosphorylation in Mgat5 /cells 1 to 5 min followingstimulation. Zap70 kinase binds to dual phosphorylated immunoreceptortyrosine-based activation motif domains of CD3ζ, and association of thelatter with Zap70 was increased in Mgat5^(−/−) compared to Mgat5^(+/+) Tcells (FIG. 3G). In summary, the Mgat5 deficiency enhancedligand-dependent TCR aggregation, and consequently, signal transductionand microfilament re-organization.

The larger size of Mgat5-modified glycans may limit the geometry andspacing of TCR clusters in the plane of the membrane (24).Alternatively, Mgat5-modified glycans may bind cell surface galectinswhich restrict TCR mobility, thus antigen-induced TCR clustering. Thegalectins are a widely expressed family of mammalian lectins defined asN-acetyllactosamine-binding proteins. The poly N-acetyllactosaminesequences preferentially added to Mgat5-modified glycans (6), enhancedthe affinity for galectin binding (FIG. 1A). Galectins bind lactosamineand lactose with dissociation constants in the 10⁻⁴ M range (7; 8), anaffinity comparable to MHC/peptide-induced oligomerization of TCRs insolution (22). Therefore, the avidity of a multivalent galectin—Mgat5glycoprotein lattice at the cell surface may be sufficient to restrictTCR clustering. To probe for the presence of galectin—glycoproteininteractions, wild type ex vivo T cells were pre-incubated with variousdisaccharides for 20 min prior to a 10 min. stimulation with anti-CD3εantibody coated beads. Pre-incubation with lactose increased TCRclustering at the bead interface and reduced TCR density elsewhere onthe cells (FIG. 4C), which is similar to the behavior of untreatedMgat5^(−/−) T cells (FIGS. 3A, B). TCR clustering was not altered bypre-incubation with the control disaccharide sucrose (FIG. 4B).Lactosamine and lactose both enhanced protein phosphorylation induced byanti-CD3ε antibody coated beads but sucrose and maltose had no effect(FIG. 4D and data not shown). Lactose did not enhance signaling inMgat5^(−/−) T cells (data not shown).

Galectin-3 was detected on the surface of naive T-cells by labeling withNHS-biotin, capture with streptavidin beads and Western blotting withanti-galectin-3 antibodies (FIG. 4E). Chemical crosslinking of the cellsurface to stabilize complexes, followed by western blotting ofgalectin-3 immunoprecipitates demonstrated that galectin-3 is associatedwith TCR complex proteins. This interaction was disrupted by eitherMgat5 deficiency or by incubating wild type T-cells with 2 mM lactose(FIG. 4E). Taken together, the data demonstrates that a multivalent cellsurface galectin-glycoprotein lattice limits TCR clustering in responseto agonist, the avidity of which is dependent upon Mgat5-modifiedglycans (FIG. 4F). The full complement of glycoproteins and lectinspresent in the T cell lattice remain to be defined but at a minimumincludes galectin-3 and the TCR complex. Others have shown thatexogenously added galectin-1 binds CD2, CD3, CD4, CD7, CD43 and CD45 andthese proteins may also participate in the lattice (25). Indeed,exogenous galectin-1 modulates T cell activation in vitro (9; 25),antagonizes TCR signaling (26), and when injected into mice, itsuppresses the pathology of EAE (27).

The gene replacement vector used to produce the Mgat5-deficient micecontained the reporter gene LacZ replacing the first exon, which wasexpressed with the same tissue-specificity as Mgat5 transcript (16).Both LacZ expression and cell-surface Mgat5-modified glycans inMgat5^(+/−) T cells, respectively, increased 48 h after stimulationdemonstrating regulation of Mgat5 by transcriptional means (FIGS. 4G,H). This suggests that Mgat5 enzyme activity and glycan production arelimiting in resting T cells, and with stimulation, increases inMgat5-modified glycans and galectins may dampen TCR sensitivity toantigen. Negative feedback by Mgat5-modified glycans on TCR sensitivityis delayed as it requires Mgat5 gene expression, which is dependent on Tcell activation status, and only indirectly on antigen concentrations.This form of slow-negative regulation governed by steady-state activityof the system is a key feature of robust and adaptive biochemicalpathways (28) and Mgat5-modified glycans may contribute this feature toT cell regulation.

Viola et at have estimated that sustained clustering of ˜8000 TCRs isrequired for T cell activation, but other molecular interactions clearlyalter this threshold. With CD28 co-stimulation, only ˜1500 TCRs arerequired (2). Co-signaling through CD28 decreases the extent of TCRclustering needed for activation predominantly by recruiting proteinkinase-enriched GM1 ganglioside rafts to the site of TCR engagement,thereby amplifying signaling (3; 5). Here it is shown that Mgat5deficiency increases the number of TCRs recruited to theantigen-presenting surface, thereby reducing the requirement for CD28co-receptor engagement This may lead to T cell activation in the absenceof CD28 co-signaling, failure of anergy and loss of immune tolerance.CD28^(−/−) mice are resistant to induction of EAE by low dose MBP, whileMgat5^(−/−) are hypersensitive, but both mutants develop clinical signsof EAE comparable to wild type littermates with high doses of MBP (29).In this regard, CD28 and Mgat5 appear to be opposing regulators of Tcell activation thresholds, and susceptibility to autoimmune disease. Insummary, Mgat5-dependent glycosylation limits agonist-induced TCRclustering by sequestering receptors in a cell surfacegalectin-glycoprotein lattice. However, the glycosylation deficiency inMgat5^(−/−) mice affects other pathways and cells types that may alsocontribute to the observed autoimmunity. Indeed, Mgat5-modified glycansalso reduce clusters of fibronectin receptors causing accelerated focaladhesion turnover in fibroblasts and tumor cells; a functionality thatmay affect leukocyte motility (16). Finally, glycosylation of Notchreceptor by Fringe, a fucose-specific β1,3GlcNAc-transferase providesanother example of regulation by differential receptor glycosylation(30). In a broad context, the results described herein suggest a generalmechanism for the regulation of receptor clustering through differentialglycosylation and interaction with cell surface lectins.

TABLE 1 Clinical observations of autoimmune encephalomyelitis (EAE)Incidence of Days with Groups (dose) EAE Peak score Onset (days) diseaseDeaths Mgat5^(+/+) (25 μg)  3/11 0.45 ± 0.24   24 ± 3.9  7.0 ± 3.9 0Mgat5^(−/−) (25 μg)  9/11# 1.82 ± 0.39* 19.8 ± 3.3* 11.5 ± 3.0* 1Mgat5^(+/+) (100 μg) 10/10  1.6 ± 0.22   25 ± 2.2 18.5 ± 2.2 0Mgat5^(−/−) (100 μg) 10/10  2.1 ± 0.34* 17.6 ± 2.9* 23.3 ± 3.5* 1Mgat5^(+/+) (500 μg) 12/12  3.0 ± 0.43  8.9 ± 1.2 27.9 ± 4.0 3Mgat5^(−/−) (500 μg) 12/12 2.83 ± 0.38  9.3 ± 0.95 27.2 ± 2.9 2

Disease severity was scored on a scale of 0-5; with 0, no illness; 1,limp tail, 2; limp tail and hindlimb weakness; 3, hindlimb paralysis; 4,forelimb weakness/paralysis and hindlimb paralysis; 5, moribund ordeath. Mean±SE of incidence, peak score, and days with disease werecalculated using the total number of mice injected per dose as thedenominator. The mean±SE for day-of-onset was determined by only usingthose mice that became sick. # Contingency test, P<0.001; and * MannWhitney test comparing genotypes for significant differences at P<0.05.

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The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. All publications, patents and patent applicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, methodologies etc.which are reported therein which might be used in connection with theinvention. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to “ahost cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

1. A cell based assay for evaluating a test compound for its ability toregulate T cell receptor clustering comprising (a) providing T cellshaving galectin-3 associated with their T cell recptor and which aredeficient in β1,6N-acetylglucosaminyltransferase V (Mgat5); (b) mixingthe T cells, and a test compound under conditions which induce receptorclustering; (c) detecting receptor clustering; and (d) comparing toreceptor clustering detected in T cells in the absence of a testcompound wherein a difference in receptor clustering indicates that thetest compound potentially regulates receptor clustering.
 2. A cell basedassay according to claim 1 wherein the cells are Mgat5^(−/−) T cells. 3.A cell based assay according to claim 1 wherein T cell receptorclustering is induced by adding an antigen presenting cell or beadcoated with an antigen or anti-TCR antibody.
 4. A cell based assayaccording to claim 3 wherein T cell receptor clustering is induced byadding anti-CD3ε antibody coated beads.